Many interferon-stimulated genes (ISGs) and other antiviral mechanisms protect humans from a large variety of viruses. Taken from a viral perspective, many ISGs represent potent barriers to viral replication. Therefore, to be evolutionarily successful, viruses must find ways to evade these host antiviral mechanisms. Indeed, multiple viruses encode specific factors that allow them to directly antagonize host antiviral proteins. The identification and characterization of such viral antagonists is thus highl relevant for understanding human susceptibility to viruses and designing new therapies and vaccines. Despite this great biomedical importance, identifying novel viral antagonists or even which host genes are antagonized by viruses has been difficult. Our previous evolution-guided functional studies have revealed that known viral antagonism of a host gene is often correlated with rapid adaptive evolution, or positive selection, in that host gene. This application seeks to greatly expand the utility of this concept by proposing that positive selection analyses can be used to predict which host antiviral proteins are targeted by unknown viral antagonists and enable identification of the previously unknown antagonists. We will focus on the IFIT (Interferon-induced with tetratricopeptide repeat) gene family, a highly interferon-induced set of broadly-acting antiviral factors that are important for host defense against several viruses. IFIT genes have no known viral antagonists. However several of them have evolved under strong positive selection, leading us to hypothesize that viruses have repeated targeted them with antagonists. We will test whether IFIT proteins are direct targets of viral antagonism by employing a combination of insights gleaned from evolutionary genetics, together with biochemistry and virology techniques. These studies will define the biochemical and functional interactions between IFIT proteins and viral antagonists and test the importance of IFIT antagonism for viral replication. Additional experiments will examine the functional consequences of IFIT evolution, and whether this defines the susceptibility of IFIT proteins to antagonism by currently circulating viruses. Finally, experimental evolution will reveal the evolutionary path that viruses take to counteract IFIT-mediated host defenses. These studies will not only shed light on the role that antagonists play in viral evasion of the IFIT- mediated antiviral response, but will also represent a potentially widely applicable strategy for the de nov identification and characterization of viral antagonists of important antiviral genes.